| DQ-Red BSA Trafficking Assay in Cultured Cells to Assess Cargo Delivery to Lysosomes
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Author:
Date:
2017-10-05
[Abstract] Lysosomes are the terminal end of the endocytic pathway having acidic environment required for active hydrolases that degrade the cargo delivered to these compartments. This process of cargo delivery and degradation by endo-lysosomes is a tightly regulated process and important for maintaining cellular homeostasis. Cargos like EGF (Epidermal Growth Factor), Dil-LDL (3,3’-Dioctadecylindocarbocyanine-Low Density Lipoprotein), Dextran, DQ-BSA (Dye Quenched-Bovine Serum Albumin) etc., are routinely used by researchers to analyze the role of various proteins in endocytic pathway. Trafficking of DQ-BSA in cells depleted of or over-expressing the gene of interest is a useful assay for identifying the role of various proteins in endocytic trafficking pathway. The protocol describes the ...
[摘要] 溶酶体是具有降解递送到这些隔室的货物的活性水解酶所需的酸性环境的内吞途径的末端。 这种内溶溶酶体的货物递送和降解过程是一个严格调节的过程，对于维持细胞体内平衡是重要的。 Cargos如EGF（表皮生长因子），Dil-LDL（3,3'-二十八碳基碳代花青 - 低密度脂蛋白），葡聚糖，DQ-BSA（染料淬灭 - 牛血清白蛋白）等常规 由研究人员用来分析各种蛋白质在内吞途径中的作用。 在缺乏或过表达感兴趣的基因的细胞中的DQ-BSA的贩运是用于鉴定各种蛋白质在内吞运输途径中的作用的有用测定。 该方案描述了可用于研究各种细胞类型的吞噬运输的DQ-Red BSA运输测定。
【背景】细胞不断地与其细胞外环境交换物质，并且在这个过程中，它们将货物内部化在质膜的囊泡中。这种内部货物被运送到早期的内体，从那里它可以通过再循环内体回到质膜或进入规范的内吞途径。一旦被注定要退化，货物就会移动到后期的内体，并最终与溶酶体融合，其中活性水解酶消化货物（Jovic等人，2010）。这些内吞室具有其内腔的特征pH。早期内体体内的pH值范围为5.9-6.8，晚期内体的pH范围为4.9-6.0，溶酶体最为酸性，pH范围为4.5-5.0（Maxfield和Yamashiro，1987）。溶酶体的酸性环境对于存在于其管腔中的水解酶和货物降解的活性是必需的（Garg等人，2011; ...
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| Efficient Isolation of Influenza Specific CTLs
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Author:
Date:
2016-06-20
[Abstract] Human antigen-specific CD8+ T-cell clones are valuable tools for dissecting CD8+ T-cell responses against antigens derived from infectious agents, cancer and self antigens. Here we describe a protocol for isolating human antigen-specific CD8+ T cells. This protocol uses surface capture of IFNγ to identify antigen responsive cells that are then cloned by single-cell sorting. Here we use CD8+ T-cell responses to influenza matrix protein (MP) as an example, but this approach can be applied to any antigen specificity.
[摘要] 人抗原特异性CD8 + T细胞克隆是用于解剖来自感染剂，癌症和自身抗原的抗原的CD8 + T细胞应答的有价值的工具。 在这里我们描述了用于分离人抗原特异性CD8 + T细胞的方案。 该协议使用IFNγ的表面捕获来鉴定抗原反应性细胞，然后通过单细胞分选克隆。 这里我们使用CD8 + T细胞对流感基质蛋白（MP）的反应作为例子，但这种方法可以应用于任何抗原特异性。
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| Mouse Macrophage Differentiation by Induction with Macrophage Colony-Stimulating Factor
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Author:
Date:
2013-09-05
[Abstract] Macrophages are differentiated from circulating blood monocytes and act as tissue-resident professional phagocytes. Macrophages function in both innate and adaptive immune systems of vertebrate animals. The cytokine macrophage colony-stimulating factor (M-CSF) is essential for the proliferation and differentiation of monocytes. Here, we described a simple method to induce the differentiation of mouse bone marrow-derived myeloid precusor cells into macrophages in the presence of M-CSF.
[摘要] 巨噬细胞与循环血液单核细胞分化，并作为组织驻留专业吞噬细胞。 巨噬细胞在脊椎动物的先天和适应性免疫系统中起作用。 细胞因子巨噬细胞集落刺激因子（M-CSF）对于单核细胞的增殖和分化是必需的。 在这里，我们描述了一种简单的方法诱导小鼠骨髓衍生的骨髓前体细胞分化为巨噬细胞在M-CSF的存在。
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